We have been examining polymorphisms in genes involved in the leptin signaling pathway to identify gene variants impacting on body composition. We are currently intensively studying a variant MC3R that is associated with adiposity in children and which appears to have functional significance for MC3R signal transduction. Children who were homozygous variant for both the polymorphisms (Thr6Lys and Val81Ile) had significantly greater fat mass and leptin compared with wild type or heterozygous children. In vitro studies subsequently found that expression was significantly lower for the double-mutant MC3R. Ongoing studies attempt to understand the mechanisms by which these sequence alterations may impact body weight. We have initiated a study comparing energy balance during adaptation to a high-fat diet in humans with double-mutant and wild type MC3R. Data from subcutaneous abdominal adipose tissue biopsies are obtained to examine fatty acid uptake and lipolysis in humans with the double-mutant receptor. We have also successfully bred and studied novel knock-in mice expressing the human wild type and human double-mutant MC3R (1). Using homozygous knock-in mouse models in which we replaced murine Mc3r with wild-type human (MC3R(hWT/hWT)) and double-mutant (C17A+G241A) human (MC3R(hDM/hDM)) MC3R, we found that MC3R(hDM/hDM) have greater weight and fat mass, increased energy intake and feeding efficiency, but reduced length and fat-free mass compared with MC3R(hWT/hWT). MC3R(hDM/hDM) mice did not have increased adipose tissue inflammatory cell infiltration or greater expression of inflammatory markers despite their greater fat mass. Serum adiponectin levels were increased in MC3R(hDM/hDM) mice and MC3R(hDM/hDM) human subjects. MC3R(hDM/hDM) bone- and adipose tissue-derived mesenchymal stem cells (MSCs) differentiated into adipocytes that accumulated more triglyceride than MC3R(hWT/hWT) MSCs. MC3R(hDM/hDM) impacted nutrient partitioning to generate increased adipose tissue that appeared metabolically healthy. These data confirmed the importance of MC3R signaling in human metabolism and suggested a previously-unrecognized role for the MC3R in adipose tissue development. We have also investigated the BDNF-TrkB pathway in regards to body mass in children. We found among patients with the WAGR (Wilms Tumor, Aniridia, Genitourinary, and Renal abnormalities) syndrome that those missing one BDNF allele (BDNF+/-) had significantly greater body mass than BDNF +/+ during childhood. We have also characterized a genetic change near the BDNF gene that appears to impact adiposity in otherwise normal children and adults (2). We examined human hypothalamic BDNF expression in association with 44 BDNF SNPs. We observed that the minor C allele of rs12291063 is associated with lower human ventromedial hypothalamic BDNF expression (p < 0.001) and greater adiposity in both adult and pediatric cohorts (p values < 0.05). We further demonstrated that the major T allele for rs12291063 possesses a binding capacity for the transcriptional regulator, heterogeneous nuclear ribonucleoprotein D0B, knockdown of which disrupts transactivation by the T allele. Binding and transactivation functions are both disrupted by substituting C for T. These findings provide a rationale for BDNF augmentation as a targeted treatment for obesity in individuals who have the rs12291063 CC genotype. We have previously found that leptin is an important predictor of weight gain in children: those with high leptin gain even more weight when followed longitudinally and identified children with hyperleptinemia and leptin receptor mutations. We have also found hyperleptinemia out of proportion with body fat mass in children with psychological loss of control over eating. Such data suggest the importance of leptin resistance as a factor stimulating weight gain and have led to recent explorations of other syndromes associated with obesity that may cause dysregulation of leptin signaling, including Bardet Biedl syndrome. Other studies are directed at understanding additional genetic, physiological, psychological, and metabolic factors that place children at-risk for undue weight gain (7-17). We have recently examined how obesity and thyroid hormone are interrelated (18) Two recent initiatives target insulin resistance. One trial studied the role of depressive symptoms in childrens insulin resistance. We have found both cross-sectional and prospective longitudinal associations between depressive symptoms and the development of insulin resistance and links between depressive symptoms and energy intake in children and adolescents. A randomized clinical trial to study the effects on insulin resistance of preventing depression in obese adolescents with a family history of type 2 diabetes is now complete (22). Another pilot study initiated in 2013 tests if short bouts of activity may improve glucose tolerance in children. In non-overweight children (10), we found that Interrupting sitting resulted in a 32% lower insulin Area Under the Curve (AUC; P < .001), 17% lower C-peptide AUC (P < .001), and 7% lower glucose AUC (P = .018) vs continuous sitting. Interrupting sedentary time with brief moderate-intensity walking thus improved short-term metabolic function in non-overweight children. These findings suggest that interrupting sedentary behavior may be a promising prevention strategy for reducing cardiometabolic risk in children. Investigations concentrating on binge eating behaviors in children suggest that such behaviors are also associated with adiposity in children, predict future weight gain in children at-risk for overweight, and predict both greater energy consumption during meals and decreased satiety after eating. The ability to consume large quantities of palatable foods, especially when coupled with decreased subsequent satiety, may play a role in the greater weight gain found in binge eating children. Using our longitudinal cohort, we have also found that the development of some aspects of metabolic syndrome in children is associated with the presence of binge eating behaviors (17). Two protocols examined efficacy of interpersonal therapy as a weight gain preventive strategy among children and adolescents who report binge eating behaviors (11). A new study will examine if retraining attentional biases away from palatable foods (6) can help children avoid weight gain. Given the rapid increase in the prevalence of obesity, the development of treatments for obesity is urgently needed. In clinical protocols, we have studied pharmacotherapeutic approaches to the control of body weight (3, 4). Metformin 1000 mg BID was studied in 100 severely overweight children (6-12y) who manifested hyperinsulinemia and insulin resistance. Compared to placebo-treated children, those randomized to metformin decreased BMI (metformin -0.910.3 vs. placebo +0.230.3 kg/m2, P=0.006), BMI-Z score (-0.110.02 vs. -0.040.02, P=0.02), and body fat mass (-1.40.7 vs. +2.10.7 kg, P<0.001) to a significantly greater extent. We concluded that metformin, added to a monthly behavioral program, significantly improved weight loss, insulin resistance, and cholesterol over a 6-month interval in severely overweight, insulin-resistant children. We also participated in a multi-site randomized-controlled trial of beloranib, a methionyl aminopeptidase 2 inhibitor, to treat the hyperphagia of patients with the Prader Willi Syndrome. We expect to initiate additional translational trials in the near future related to modulation of the leptin signaling pathway. Finally, we have initiated studies to examine the hypothesis that administration of colchicine can decrease NLRP3-activated inflammation and improve obesity-related metabolic dysregulation.